the plant journal ~"

The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato

Eduardo O. Leidi1, Verónica Barragán" Lourdes Rubia2, Abdelaziz EI-Hamdaoui' , M. Teresa Ruiz1, Beatriz Cubero' , José A. Fernández2

, Ray A. Bressan3, Paul M. HasegawaJ

, Francisco J . Quintero 1 and José M. Pardo"·

, .

1 Instituto de Recursos Naturales y Agrobiología (lRNASE), Consejo Superio r de Investigaciones Científicas. Reina Me rcedes,

lO. Sevilla - 41012, Spain,

20 epartamento de Biología Vegetal, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, Málaga - 29071, Spain, and

3Center far Plan t Envíronmental Stress Physiofogy, 625 Agriculture Mal! Orive, Horticulture Bldg, Purdue University,

Wesl LalayeNe, IN 47907·20 10, USA

SUMMARY

NHX-type antiporters in the tonoplast have been reported to ¡ncrease the salt tolerance of various plants species, and are thought to mediate the compartmentation of Na+ in vacuo les. However, all isoforms characterized so far catalyze both Na+/H+ and K+/H+ exchange. Here, we show that AtNHX1 has a critical involvement in the subcellular partitioning of K+, which in turn aftects plant K+ nutrition and Na+ tolerance. Transgenic tomato plants overexpressing AtNHX1 had larger K+ vacuolar pools in all growth conditions tested. but no consistent enhancement of Na+ accumulation was observed under salt stress. Plants overexpressing AtNHX1 have a greater capacity to retain intracellular K+ and to withstand salt-shock. Under K+-limiting conditions, greater K+ compartmentation in the vacuole occurred at the expense of the cytosolic K+ pool. which was lower in transgenic plants. This caused the early activation ofthe high-affinity K+ uptake system. enhanced K+ uptake by roots, and increased the K+ content in plant tissues and the xylem sap of transformed plants. Our results strongly suggest that NHX proteins are likely candidates for the W-linked K+ transport that 15 thought to facilitate active K+ uptake at the tonoplast. and the partitioning of K+ between vacuo le and cytosol.

Keywords: NHX, vacuole. potassium transport, salt tolerance, tomato.

INTRODUCTION

A critical process in the sa lt t ole rance of plants is the eom ­

partmentation of Na+ in vacuo les, which prov ides an effi eíent

mee hanism to ave rt t he delete ri ous effeets of Na+ in the

eytoso l, wh ile mediat ing vacuo lar osmot ie adjustment that

l ae ilitates wate r uptake into ce lis (Blumwald el al., 2000). The

NHX proteins were o riginally deseribed as Na+/H+ antiport­

ers located in the tonop last, driving Na+ aecu mulation into the vacuo le, and eonfe rring salt toleranee (Apse et al., 1999; Gaxiola et al., 1999; Quintero et al., 2000). Howeve r, in sp ite

of numerous reports of imp roved ha lotole ranee by the

overexpression of NHX·type exehangers in various plant

speeies, the meehanism underlying the enhaneement of

salinity toleranee by these t ransporters ls nol yet clear

(Tester and Oavenpo rt, 2003; Pardo el al., 2006). Severa l

reports fai led to find the anticipated eorre lation between

increased salt tolerance and enhanced accumulation of Na+

by NHX proteins (Ohta el al., 2002; Fukuda el al., 2004; Wu

et al., 2009), whereas others found greater K+ eontents,

rather than greater Na+ contents, in tissues of the transgenie

plants (Xue et al., 2004; Wu et al., 2005; Zhao el al., 2006; Rod riguez-Rosa les et al., 2008). AII plant NHX-type t ran so

po rters cha raete rized to date can be elassified into two

groups: elass I and elass 11 (Brett el al., 2005; Pardo el al.,

2006). Proteins of class I localize to vacuo les and eatalyze

Na+/H+ and K+/W exchanges in vitra, whereas the endoso­

mal (non-vacuo lar) class- II proteins may show a preference

fo r K+ over Na+ as the substrate (Venema et al., 2002, 2003;

Apse el al., 2003; Rodriguez-Rosa les el al., 2008). Bioehem­

ical studies have established that AtNHX1, a class- I protein,

catalyzes both Na+/W and K+/W exehange with simila r

affinity (Venema el al., 2002; Apse el al., 2003; Yam aguehi et al., 2005). Moreover, bind ing of the ealmodu lin-l ike pro­

tein AtCa M15 of Arabidapsis thaliana to the C terminus of

AtNHX1 lowered the maxim al ion-exehange rate (Vmax) of

the Na+jH+ exchange activity in isolated vacuo les, whereas

the Vmax of the K+jH+ exchange activity was not significantly

altered, thus leading to a decrease in the Na+ : K+ transport

ratio (Yamaguchi et al., 2005). Indirect evidence from

resto red tolerance of yeast nhx1 mutants to NaCI and KCI

suggested that the vacuolar proteins OsNHX1 from Oryza sativa Irieel, and InNHX1 and InNHX2 from Ipomoea ni! (Morning glory), also have dual Na+ and K+ specificity

IFukuda et al., 2004; Ohnishi et al., 20051. Thus, insuffieient Na+-K+ discrimination brings into question the role of

vacuo lar NHX1-like proteins as effective Na+ scavengers in planta, as the cytosolic K+ concentration will most likely ex­

ceed that of Na+, even under salinity stress (Carden et al.,

2003; Tester and Davenport, 20031. Cytosolie Na" eoneen­

trations of 10-30 mM have been estimated in the rootcells of salt-treated glycophytes by X-ray microanalysis and

ion-sensitive electrodes, whereas cytosolic K+ will often

remain aboye the 60 mM level (Hasegawa et al., 2000; Carden et al., 20031.

Besides their role in salinity tolerance, N HX protei ns have

been implicated in the control of vacuolar pH in petal cells

of Ipomoea IFukuda-Tanaka et al., 2000; Yamaguehi et al., 2001; Yoshida et al., 20051, and of leaf eell expansion in

Arabidopsis IApse et al., 20031. The partieipation of NHX proteins in these pro ces ses is most likelyto be related to K+j

H+ exchange, as Na+ concentrations in the cytosol of plant

cells remain low in the absence of salinity stress. Vacuoles

occupy most of the intracellular volume in many plant cells and are the main cellular resetvoir for K+. Changes in tissue

K+ concentration are largely a reflection of the dynamics of

the vacuo lar pool. By contrast, cytosolic K+ concentrations

are tightly regulated through the integrated regulation of K+

uptake and efflux at the plasma membrane, and K+ import

and export at the tonoplast ILeigh, 20011. Cytosolie K" concentration is thought to decline only when vacuolar K+

has been depleted IWalker et al., 19961. Conversely, surplus K+ is placed into the vacuole to maintain cytosolic K+ within

narrow limits, independently of K+ abundance in the growth

medium. At the usual range of tonoplast membrane potential (20-40 mV, positive inside) and cytosolic K+ activ­

ity (80 mM), transport of K+ into the vacuole in K+-replete

cells occurs against the electrochemical gradient, and must

be energized IWalker et al., 1996; Martinoia et al., 20001. Two

mechanisms have been proposed for active accumulation of K+ in the vacuole (Leigh, 2001). One is the vacuolar

pyrophosphatase that may couple H+ and K+ pumping into

the vacuo le, but this has not been confirmed experimentally. A K+jH+ antiporter energized by the pH gradient across the

tonoplast has also been suggested to control vacuo lar K+

accumulation, albeit the molecular identity remains elusive

IWalker et al., 1996; Carden et al., 20031. Vaeuolar N HX-type

exchangers could se tve this critical function in plant cells.

To obtain further insights into the physiological role of

NHX proteins, we undertook a detailed characterization of

the ion relations of transgenic tomato overexpressing the

vacuolar antiporter AtNHX1 of A. thaliana under different supplemental K+ and Na+ regimes. Our results show a

critical involvement of AtNHX1 in the subcellular partition­ing of K+ that, in turn, affects plant K+ nutrition and Na+

tolerance.

RESULTS

Phenotype 01 transgenic lines

The eDNA eorresponding to the Arabidopsis AtNHXl gene

IAt5g271501 IYokoi et al., 20021 was expressed under the cauliflower mosaic virus (CaMV) 355 gene promoter in

transgenic tomato plants (50lanum Iycopersicum cv.

MicroTom). Transgene expression in kanamycin-resistant

plants was analyzed by northern blotting and RT-PCR with

AtNHX1-specific pro bes and primers, respectively. Four

independent homozygous lines IN-367, -374, -376 and -3801

were selected for further study beca use of they showed

aeeumulation of the AtNHXl transeript IFigure 511. The

abundanee of the AtNHXl mRNA in these transgenie plants

was not affeeted by salt stress 15 h in 150 mM NaCI; Fig­

ure 511. Among the transgenie lines tested, line N-367 had

the highest expression level of AtNHX1. Western blotting of

microsomal fractions from line N-367 probed with poly­

clonal antibodies raised against AtNHX1 demonstrated the

accumulation of the Arabidopsis protein (Figure 1a). Tono­

plast vesicles from the roots (Figure 1b) and leaves (data not

shown) of line N-367 showed greater Na+jH+ and K+jH+

exchange activity than vesicles from control plants. Both the exchange of Na+jH+ and K+jH+ increased twofold in trans­

genic plants over the background of the endogenous anti­porter activity in tomato (Figure 1c). No significant

difference was found in vacuolar ATPase activity, and in the

magnitude of inside-acid pH gradients performed in tono­

plast vesicles from control and transgenic plants (Figure 1c).

The commensurate gain in Na+jH+ and K+jH+ exchanging

capacities in tonoplast membranes of the N-367 plants is in agreement with the low Na+-K+ selectivity of AtNHX1, pre­

viously determined in artificial proteoliposomes and Ara­

bidopsis vacuo les IVenema et al., 2002; Apse et al., 20031. Rates of Na+jH+ and K+jH+ exchange in the vacuoles of

control plants were subtracted from the rates in transgenic

plants and fitted to Michaelis-Menten kinetics. The Km of AtNHX1 protein was estimated at 22 ± 12.8 mM for Na+ and

20.9 ± 14.7 mM for K+. Vmax was also similar for both cations

[116 ± 20 for Na" and 96.5 ± 19.9 for K"; measured as the

relative change in fluorescence (fj,FjFmax ) mg-1 min-1].

Together, these results demonstrate that transgenic lines

had a greater number of active NHX1-type transporters than control plants, and that they had low Na+-K+ selectivity.

Overexpression of AtNHX1 has been shown to convey salt tolerance to tomato plants (cv. Moneymaker) (Zhang and

Blumwald, 2001). Microtomato plants overexpressing

(a) KDa

50~

(b)350

300

e ·E 250

en E 200 x

J. 150 ....... u.. <l 100

50

O

(e) 350

300

e 250 ·E E 200

x 150 E u.. ....... 100 u.. <l

50

O

N T M

O 50 100 150

NaCl or KCI (mM)

b b

V-ATPase Na+/H+ K+/H+

Figure 1. Na+/W and K+/W exchange in tonoplast vesicles.

C

e

200

(a) Mierosomal proteins (30 ~Lg) from wild-type (T) and N·367 transgenie plants (N) were probed with antibodies against the AtNHX1 protein. C, 100 ng of purified AtNHX1; M, molecular size markers. Left, chemiluminiscence after western blotting; right, Coomassie staining. (b) ATP-dependent inside·acid pH gradients were preformed in tonoplast vesieles isolated from roots of wild-type (0, D) and N-367 transgenies (e, . ), and the rate of dissipation was measured after the addition of NaCI (eircles) or KCI (squares) salts at the indieated final eoneentrations. Exchange aetivity is expressed as arbitrary units (fluoreseenee reeovery per minute and milligram of protein). Curves were fitted by the least·squares method. (e) Vaeuolar W-ATPase and Na+-K+/W exehange aetivities. Inside·aeid pH gradients were established in tonoplast vesicles purified from the roots of wild-type (white bars) and N-367 transgenie plants (black bars) after the addition of ATP. Proton pumping by the V-ATPase was caleulated from initial rates of fluorescenee decline. Once fluoreseence was stabilized, 100 mM of NaCI or KCI salts were added, and fluoreseenee reeovery indicating eation/ proton exehange was monitored for 2 mino Aetivities are expressed in arbitrary units as the relative change in fluoreseence (!1F/Fmax) per minute and milligram of membrane protein. Values are means of three independent biologieal replieates, different to those shown in (b). Letters a and b on top of pair·wise eomparisons indieate: a, no significant differenee, by the least signifieant differenee test; or b, statistieally different values at P < 0.05.

AtNHX1 were more salt tolerant than control plants when the stress treatment was applied as salt-shock (Figure 2). By

contrast, when salinity was increased stepwise at 25-mM

(b) 0.3 b

,-.9 e lA e a

~ 8 ,-.

Ci 'L 1.2 -- 7 e - í ..c CI -E. 1.0 .~0.2 CI

6 al -- CI s: -5 g 0.8 >- e ... al 4 "E 0.6 "C -e al 00.1 o 3 -(J e 004 o ... o ..c al 2 (J en - e 0.2 ca 1 s: .E

0.0 O 0.0 K Na

Figure 2. Salt tolerance and K+ deficieney. (a) Wild·type and transgenie N·367 plants were treated with 150 mM Na,SO. for 6 days, and werethen allowed to recoverfor4 days withoutsalt. Shown are three representative plants from the eontrols (left) and from line N-367 (right). (b) Shoot dry weight. water content, and K+ and Na+ eontents of wild-type (white bars) and N·367 transgenic plants (black bars). treated as deseribed in (a). Values are means and SDs of eight plants. Letters a, b and con top of pairo wise comparisons indieate: a, no significant difference by the least signifieant differenee test; b, statistieally different val ues between genotypes at P < 0.05; and e, statistically different values between genotypes at P < 0.01. (e) Symptoms of K+ defieieney. Plants were grown for 2 weeks in hydroponie culture eontaining 0.1 mM K+, without renewal of the hydroponic solution. Representative leaves of control (Ieft) and transgenie (right) plants are depieted. The eoneentrations of K+ were 42.8 mM and 70.2 mM in leaves of control and N·367 plants, respeetively, at the end of the experiment.

increments twice a day, to reach final concentrations of 75 or

150 mM NaCI, plant growth was similarly inhibited in control

and all transgenic lines tested (Tables 1 and 81). In these

Table 1 Shoot growth and K+ and Na+ concentrations in wild-type and four transgenic lines under moderate salinity stress. Wild-type (WT) and plants from four independent transgenic lines (N-367 through N-380) were grown in hydroponic media with (75 and 150 mM) or without supplemental NaCI. After 3 weeks, plants were harvested, the shoots were weighted and their mineral concentrations were determined by atomic absorption spectrometry. Values are in grams of fresh weight per plant (FW) and millimolar concentrations (K+ and Na+). Means followed by different letters were statistically different at P < 0.05 (Ieast significant difference test)

o 75

NaCI (mM) FW K+ Na+ FW

WT 1.76a 103.7a 3.2a 1.48a

N-367 2.88b 142.6b 3.4" 1.53" N-374 1.48a 171 .0c 4.1a 1.10a

N-376 1.43a 169.0c 4.1 a 1.19a

N-380 2.31 c 145.6b 3.7a 1.31 a

conditions, both control and transgenic plants completed their life cycle and set fruits after 8 weeks (Table 81). Notably, no consistent enhancement of Na+ accumulation was observed in the AtNHX1-overexpressing transgenic lines under all saline regimes tested, including the salt-shock treatments to which the transgenic lines showed greater tolerance than control plants (Figure 2; Tables 1 and 81). Instead, K+ contents were consistently greater in transgenic lines under nearly all growth conditions analyzed, including the absence of saline stress and varying K+ availability (Tables 82 and 53). To test how this differential accumula­tion of K+ affected the mineral nutrition of transgenic lines, plants were transferred to low K+ concentration (0.1 mM K+)

hydroponic medium and grown until K+ was nearly depleted in the medium. Transgenic plants exhibited earlier develop­ment of K+-deficiency symptoms (crinkled upper leaves, yellowish bronze mottling and chlorosis along the leaf margin; Figure 2c) relative to control plants, despite the fact that leaves of transgenic plants contained greater total K+ concentrations than those of control plants at the end of the experiment (70.2 mM in N-367 and 42.8 mM in control plants) . Together, these results indicate that AtNHX1 over­expression has a greater impact on K+ homeostasis than on Na+ accumulation under the growth regimes tested. More­over, these results suggestthat the greater K+ levels may not be physiologically accessible to transgenic plants growing in a K+-limiting environment, presumably because of enhanced compartmentation into the vacuole at the expense of the cytosolic K+ pool.

K+ uptake

Consistent with the hypothesis of the reduced cytosolic availability of K+ in the AtNHX1 transgenic lines under K+ limitation, roots of line N-367 that had been starved for K+ for 48 (data not shown) or 96 h (Figure 3a) showed greater rates of Rb+ uptake (a tracer for K+) than control roots. The rate of Rb+ uptake was higher in the transgenic plants at all con­centrations tested (10 ¡.IM-10 mM range) . Fitting these data to Michaelis-Menten kinetics indicated similar apparent affinities for Rb+ in control and N-367 plants (Km 58.4 and

40.8a 93.4a

56.9b 103.9" 61 .4b 101 .8" 48.8a 145.8b

54.2b 117.1a

(a) 6

í e 'E 5

a: 4

í 0>3 'O E 3 2 + .o tI:

150

FW K+

0.83a 19.3a

0.61 b 34.7b

0.36c 33.5b

0.42c 24.4a

0.67"b 36.4b

O+----r---,----~---r--~ O

(b) 0.3

~ g + ::.:::

0.2

0.1

Figure 3. Rb+ and K+ uptake.

2 3 4 5 Rb+ (mM)

5 10 15 20 Time (h)

Na+

252.7a

221.7" 177.5" 225.7a

171 .2a

la) Plants were starved lor 96 h in K+-Iree hydroponic solution, and were then translerred to Iresh media with 0.01, 0.1, 0.5,1 and 5 mM RbCI. Alter 10 min, roots 01 wild-type 10 ) and N-367 transgenics le) were washed, and the Rb+ content was determined by AAS. Values are averages 01 three biological replicas. Uptake rates were litted to Michaelis- Menten kinetics by using non linear regression. lb) Time-course 01 K+ withdrawal Irom hydroponic solution with 0.25 mM KCI, recorded with a K+-specilic electrode, in mature wild-type plants 10 ) and N-367 transgenics le).

62.5 ~LM, respectively), but twice the Vmax in the transgenics relative to control plants (4.48 versus 2.26 ¡.Imol Rb+ g-1

min-1), suggesting that N-367 plants had more active root Rb+ (K+) transporters than control plants, i.e. transgenic

plants were quantitatively but not qualitatively more responsive to K+ starvation. Similar results were obtained with plants 01 transgenic line N-380 (data not shown), sup­porting that this phenotype arose from the overexpression 01 AtNHX1. Equivalent results were obtained when K· up­take was measured by monitoring K+ withdrawal from the hydroponic solution with a K'¡' -specific electro de. As shown in Figure 3(b), rates of K+ uptake were greater in plants ofthe transgenic line relative to the control line. Consistent with enhanced root K+ uptake, xylem sap collected from excised roots of transgenic fine N·367 plants grown in 4 mM K+ contained much greater K+ concentrations than sap from control roots (32.9 and 8.4 mM, respectively). Non-starved plants of various independent transgenic lines in standard growth conditions (4 mM K+) showed significantly higher K+ concentrations in leaves and roots than control plants (Ta­ble 82). Together, these results demonstrate that plants overexpressing AtNHX1 take up K+ more rapidly, and are more responsive to K';" deprivation, than control plants.

High-affinity K+-uptake systems mediate K+ nutrition at low external concentrations, and are induced by K+ depri­vation (Rodriguez-Navarro, 2000). The HAK5 gene is induced in K+·starved tomato plants, and its express'lon pattern parallels the presence of high-affinity K+ uptake in tomato roots, suggesting that this gene encodes a component of K+ uptake in the micromolar range of K+ concentrations (Nieves-Cordones et al., 2007, 2008). Control and N-367 plants growing in 4 mM K+ were transferred to K+-free hydroponic solution, and the express ion of HAK5 was analyzed by RT-PCR in roots after 48 and 96 h 01 starvation. As depicted in Figure 4(a), HAK5 expression was induced earlier in the transgenic plants than in control s, indicating that plants overexpressing AtNHX1 sen sed K+ deficiency belore the controls. Induction 01 HAK5 in wild-type plants occurred only after several days of K+ starvation (Nieves­Cordones et al., 2007; data not shown). In a control exper­iment, Rb+ uptake by roots of plants starved as described above were transferred to a fresh hydroponic culture containing either lOor 100 ~M RbCI, and the rate 01 Rb+ accumulation in roots was determined by atomic absorption spectrometry. As shown in Figure 4(b), transgenic plants had a greater capacity for Rb+ uptake in the micromolar range that was statistically significant at 96 h of starvation, in accordance with the upregulation of gene HAK5.

Vacuolar compartmentation of K+ and Na+

A combination of scanning electron microscopy and energy dispersive X-ray microanalysis (8EM-EDX) was used on

frozen sections to measure the vacuolar ion contents in various leaf and root cell types of salt-treated tomato plants (Table 2). Although this technique does not enable the quantification of absolute ion concentrations, changes in the relative abundance of various mineral elements can be assessed. As net K" concentration in planttissues is largely a

(a) WT N-367

+K 48h 96h +K 48h 96h ------------

===HAK5 ----'*----- .... --(b) B

7

1: 6

~b 5

'04 E 33 :b 2 ti:

1

o 10 IJM 100 IJM 10 IJM 100 IJM

48h 96h

(e) Control Salt

WT NHX WT NHX -- ------R L R L R L R L

HAK5 Actin

Figure 4. HAK5 expression induced by K+ starvation.

Actin

(al Plants growing in 4 mM K+ were transferred to K+·free medium for 48 or 96 h, or were kept in the presence of K+ I+Kl until all samples were collected. Shown is the amplification (25 cyc!esl by RT·peR of HAK5in rools of duplicate biological samples. The actin TOM41 gene was used as an internal standard. (b) Root Rb+ uptake in wild·type (white barsl and N·367 plants (black bars) after being transferred to K+·free hydroponic solution for 48 and 96 h, and then to the same solution supplemented with lOor 100 11M Rbel for 10 mino Roots were excised and the Rb+ contentwas determined by atomic absorption speetrophotomctry. Va[ues are means and SEs of Rb+ uptake per gram of fresh weight of roots of triplicate biological samplcs. Within treatments, the letters a and b on top of pair,wise comparisons indicale: a, no significant differcnce by the least signifieant difference test; or b, statistically different values at P <: 0.05. (e) Wild·lype (WTI and N·367 (NHX) plants were transferred lo fresh hydroponic media with (salt) or without (control) 150 mM SO,Na2' and were then gtown for 2, 4 and 6 days. Shown is the amplification (35 cycles) by RT· peR of HAK5in root (R) and leaf (L) samples after 2 days in salt. HAK5 could not be amplified from samples corresponding to 4 and 6 days of salt treatmenl. The actin TOM41 gene was used as ¡he internal standard.

reflection of the vacuolar pool, the actual K+ and Na+ con­

centrations in the remaining parts of plants processed for SEM-EDX were determined by atomic absorption spec­trometry to ensure that SEM-EDX results correlated with tissue ion contents. Linear correlations between actual ele­ment concentrations (mM) and EDX recordings (counts) were statistically significant (K+, r = 0.68, P < 0.05; Na·, r = 0.85, P < 0.011. Vacuo les 01 leal mesophyll cells 01 N-367 plants showed consistently greater signals for K+ ions in

Table 2 Scanning eleGtron microscopy and energy dispersive X-ray microanalysis {SEM-EDX) ofthe mineral content ofvacuoles. Wild­type {WT) and N-367 transgenic plants were grown hydroponically in nutrient solution with 75 or 150 mM NaCI for 3 weeks. Relative contents of K+ and Na+ in vacuoles of leaf mesophyll parenchyma, root cortex and vascular cylinder ce lis were determined by EDX analysis. Samples from both lines were processed simultaneously for SEM-EDX analysis, and the quantitation was performed on between six and nine cells from each tissue type and plant. Values presented are percentages of the total counts. Within pairwise comparisons, means followed by different letters were statistically different at P < 0.05 {Ieast significant difference test)

K'

Leaf

75 mM NaCI Palisade

WT 0.023

N-367 0.41 b

Lagunar

WT 0.093

N-367 0.36b

150 mM NaCI Palisade

WT 0.153

N-367 0.24b

Lagunar

WT 0.133

N-367 0.44b

Na'

0.63" 0.83"

0.233

0.83"

0.673

0.73"

K' Na'

Roo!

Cortex

0.323 0.363

0.48' 0.623

Vascular cylinder

0.173 0.863

0.24b 1.11 3

Cortex

0.863 1.243

1.953 0.63b

Vascular cylinder

0.363 1.603

2.99b 1.0s"

SEM-EDXthan control plants in two different saline regimes. By contrast, Na+ content in leaf vacuoles did not show sig­

nificant differences among lines. Vacuolar contents in the root cortex and vascular cylinder of plants grown in 75 or 150 mM NaCI showed a similar pattern, with greater K+

accumulation in cells of AtNHX1-overexpressing plants than in controls, al beit mean val ues were not statistically different in cortical cells. At 150 mM NaCI, both the cortical and vas­cular cells of N-367 plants showed reduced vacuo lar Na+ contents relative to controls. Of the eight pair-wise com­parisons of Na+ vacuo lar contents in Table 2, in only one instance (cortical cells at 75 mM NaCI) was the vacuolar

content of Na+ greater in the transgenics. These results, together with the total Na+ concentrations determined in whole plants as described aboye, indicate that overexpres­

sion 01 AtNHX1 did not appreeiably enhanee the eapaeity 01 Na+ sequestration into vacuo les.

The sequestration of ions in the vacuo lar lumen is accompanied by the concurrent synthesis and accumulation 01 compatible salutes in the eytosol (Hasegawa el al., 20001. Therefore, the concentrations of free sugars and of the

amino acid proline were determined in control and trans­genie plants 01 line N-367 with and without 150 mM NaCI. Sugars were significantly greater (P < 0.05) in leaf and roots

Table 3 Accumu lation of compatible salutes. Leaf and root concen­trations of proline and free sugars, and the osmotic potentials, of plants grown in nutrient solution in the absence or presence of 150 mM NaCI for 30 days. The concentration of sugars was deter­mined colorimetrically bythe phenol-sulfuric method using glucose as the standard. Proline was determined by HPLC. The concentra­tions of sugars and proline are given in ¡.tmole per gram of fresh weight. The osmotic potential was measured with a TruPsi Water Potential Meter, and is given in mPa. Within pairwise comparisons of plant lines, meansfollowed by letlers indicate statistical significance: 3p < 0.05 or bp < 0.10, based on a least significant difference test

Line and Proline treatment leaf

No salt WT 6.55 N-367 6.26

150 mM NaCI WT 7.7 N-367 10.23

Proline Sugars Sugars 'Yo root leaf root leaf

0.51 60.5 12.1 -1.01 0.73 98.23 16.53 -1.09

2.28 28.0 10.1 -1.52 3.48b 33.83 14.2 -1.52

'1'0 root

-0.60 -0.72

-1.12 -1.01

of transgenic plants in the absence of salt stress (Table 3).

The greatest increase (62%) was found in leaves of trans­

genic plants relative to control leaves. After 30 days in salt, transgenic plants still had slightly greater concentrations of free sugars in leaves and in roots, albeit the differences in roots were not statistically different. Proline concentrations,

which were not different among plant lines without salt stress, were significantly greater in leaves and roots of the transgenic plants under salt stress (Table 3). As expected,

the osmotic potentials in both control and transgenic plants

were more negative under salt stress than in regular growth medium. However, the organic (proline and sugars) and inorganic (K+ and Na+) solute concentration differences

among plant lines did not result in different osmotic potentials of the transgenic plants relative to controls (Table 31.

Next, we tested whether the differential upregulation of HAK5 in the transgenic lines also occurred under salinity stress. Enhanced express ion of HAK5 could, theoretically, contribute to better Na+-K+ discrimination in saline media

(Rodriguez-Navarro, 20001. Control and N-367 plants were

subjected to salt-shock with 150 mM Na2S04 (i.e. similar conditions to the experiment shown in Figure 2) for 2,4 and

6 days. As depicted in Figure 4c, roots of transgenic plants

had a slightly higher HAK5 mRNA level belare stress treatment. However, the low mRNA levels in both plant lines (RT-PCR needed 35 amplilication eyeles instead olthe 25 cycles used in panel A) were further repressed by salinity

stress. Salt-induced repression of HAK5has been previously reparted (Nieves-Cordones el al., 20081.

Cytosolic K+ activity in K+ -limited plants

Presumably, the enhaneed aetivity 01 AtNHX1 in the tono­

plast of transgenic tomato plants promoted the accumula­tion of K+ into the vacuole at the expense of a diminishing

cytosolic K+ pool. To test this hypothesis, electrophysiolog­

ical measurements were performed to assess K+ activity in

the cytosol oftransgenic plants. First, epidermal root cells of

K+-limited (0.1 mM KCI) seedlings of wild-type and N-367

lines were probed using single electrodes. No significant

differences were observed in the plasma membrane poten­

tials (Em ) between seedlings of the wild type and line N-367

(-168 ± 10 and -163 ± 11 mV, respectively)(Figure 5). After

the addition of 1 mM NaCN and 1 mM salicylhydroxamic

acid (SHAM) to the assay media, to inhibit ATP synthesis and

W pumping, new resting membrane potentials (apparent

diffusion potentials, ED) were reached (Figure 5). Notably, ED

values were lower in the wild type (-100 ± 7 mV; n = 5) than

(a) -200

NaCN-SHAM .. -150

;;-.s -100

",E

-50

... wash

10 min

(b) -200

NaCN-SHAM .. -150

;;-E ...... -100

",E

-50

10min 4wash

KCI (mM) (e) 0.1 10 100

O

-20

-40 ;;-.s -60

",0 -80

-100

-120

Figure 5. Diffusion potentials IEol at different external K+ eoneentrations. la) Representative reeording of the membrane potentials in single epidermal

root eells of wild-type plants. Alter the addition of 1 mM NaCN and 1 mM SHAM, plasma membranes depolarized until a new resting membrane potential was reaehed. Then, the external K+ eoneentration was inereased

step-wise, as indieated by the arrow heads; wash indieates the onset of an NaCN-SHAM wash.

lb) Representative reeording of transgenie N-367 seedlings. le) Diffusion potentials IED) at different external K+ eoneentrations measured

in epidermal root eells of wild-type lO) and N-367 transgenie seedlings le). Values are means of five independent experiments, similar to those shown in la) and lb).

in N-367 plants (-77 ± 14 mV; n = 5). Under resting condi­

tions in ATP-depleted cells, cytosolic K+ serves as a charge

balance that counteracts membrane depolarization in

response to the increase in the external K+ concentration,

until equilibrium is reached (ED = O) (Mith6fer et al., 2005).

The less negative ED (j.e. more depolarized) value in trans­

genic N-367 plants suggests a lower cytosolic K+ concen­

tration relative to control plants. As expected, ED values

showed a strong dependency on external K+, the most

permeable ion in plant cell membranes, in both transgenic

and wild-type cells (Figure 5a,b). The slopes of changes in

ED in response to external K+ were similar (25-30 mV for a

10-fold increase in extracellular K+), indicating that the

permeability of the plasma membrane to K+ did not differ

among plant lines (Figure 5c). However, root cells of N-367

plants showed less negative ED values than wild-type cells

over all external K+ concentrations tested (Figure 5c), indi­

cating that K+ activity in the cytosol oftransgenic plants was

diminished (Mith6fer et al., 2005). On the other hand, similar

increases in external Na+ concentrations elicited equivalent

depolarization of membranes of only 10 mV in both wild­

type and N-367 epidermal root cells (data not shown).

To measure the actual cytosolic K+ activity (K\yt), double­

barreled K+-selective microelectrodes were used to impale

epidermal root cells of control and transgenic plants grown

in 0.1 mM KCI (Figure 6). K+cyt activities were calculated from

microelectrode calibration curves (slopes were close to

51 mV/pK+). There were no significant differences between

Em measurements in transgenic and control plants

(-168 ± 3 mV in the wild type and -165 ± 5 mV in N-367;

n = 3) when the double-barreled K+-selective microelec­

trodes were used, and the Em values were similar to those

registered with single-barrel microelectrodes, as described

aboye. By contrast, K\yt was significantly different between

genotypes (Figure 6). The K\yt determined in impaled wild­

type seedlings was 98 ± 1.3 mM, but was only half that, i.e.

55 ± 2.2 mM (n = 3), in the N-367 seedlings. Together, these

results demonstrate that overexpression of AtNHX1 reduced

K+ activity in the cytosol of transgenics plants under limiting

(0.1 mM) levels of external K+.

DISCUSSION

Based on biochemical and physiological data, a critical role

for tonoplast Na+/W antiporters in minimizing cytoplasmic

Na+ concentration through the sequestration of this ion in

vacuoles has been proposed (Blumwald et al., 2000). Thus

far, the only Na+-K+/W antiporters known to localize to the

tonoplast of plants belong to the NHX family. Overexpres­

sion of NHX proteins from various sources have been shown

to increase salt tolerance in a range of plant species (Pardo

et al., 2006; Munns and Tester, 2008). However, only a few of

these studies have addressed the ion relations of the trans­

genics overexpressing NHX proteins. Results have been

disparate, ranging from enhanced Na+ or K+ accumulation in

500 (a) 500 (b)

100 T 100 T 'i' .s

E- 10 10 + ~

1 1

-200 (e) -200 (d) T

-150 -150

> E 'E-lOO l!.i

-100

-50 -50

O ~ 10 min

O

Figure 6. Simultaneous measurement of membrane potential and cytosolic K+ concentration, Arrowheads ind'lcatethe addition of 1 mM NaCN and 1 mM SHAM lo the assay medium to inhibir respiration and suppress the activityof the plasma membrane prolen pump. Membrane potentials depolarized to diffusion level, whereas cytasolicK+ activity remained unchanged. Shown are representative traces of three raplícates. (al Cytosolic K+ activity was determinad with a double-barreled electrode in single epidermal roo! cells af the wild type. (b) Cytosolic K+ activity in transgenic N·361 seedlings. (e) The membrana potential was recordad in a wild·type seedling with the

double·barreled electrade. (d) Membrane potential recorded in transgenic N·367 seedling.

the transgenics to no significant differences with controls (reviewed by Pardo el al., 20061. Moreover, it should be noted that K+ and Na+ ions are unevenly distributed between

the vacuole and cytoplasm of plant cells, and none ofthese studies addressed the number of ions compartmentalized in the vacuoles relative to their net tissue contento We show here that the expression ofthe vacuo lar Na+-K+/H+ antiporter AtNHX1 in tomato increased the K+ concentration in leaves and roots in all growth conditíons tested, with negligible impact on the capacity for Na+ sequestration inside vacuo les when plants were subjected to salinity stress. The larger vacuolar K+ pools detected in transgenic plants, together with the reduced K+ cytosolic activity under K+ limitation, indicate that K+ was preferentially accumulated in vacuoles even at the expense of depleting the cytosolic compartment when external K+ was low. Diminished cytosolic K+ activity in K+-starved transgenic plants resulted in the earlier devel­opment of K+ nutríent deficiency symptoms, faster tran­

scriptional activation of HAK5 and greater K+ uptake by roots. Thus, transgenic plants intensified the response to K+ starvation despite the greater overall tissue K+ content ofthe

transgenics, Vacuoles are K+ reservoirs that ensure an ade­quate supply to the cytosol in a low-K+ environment (Leigh,

2001). In barley roots, cytosolic K+ concentration declined only when vacuolar K+ concentration decreased to values around 20 mM, or when the whole-root K+ concentration fell below 25 mM (Walker el al., 1996; Leigh, 20011. Although we could not measure the precise concentration of K+ in the vacuoles of plants beca use of the semi-quantitative nature of EDX analyses, the relative K+ contents were significantly greater in the transgenics than in control plants in the cell types tested (Table 21. Electrophysiological studies suggest the suitability 01 tonoplast K·¡W antiporters for the homeo­static regulation of cytosolic K+ when ce lis are provided with surplus concentrations of K+ (Walker et al., 1996), Assuming

a tonoplast membrane potential of 30 mV (positive inside), the vacuolar K+ concentration would be 20 mM at equilib­rium with the cytosol if the cytosolic concentration was ",,100 mM. Any further increase of the vacuolar concentra­tion would necessitate energized K+ transport across the tonoplast, presumably mediated by K+/W antiporter activity (Walker el al., 19961. Our data strongly support the notion that N HX-type proteins serve this function. Recently, the K+/ W exchange activity of the ItNHX1 protein has been impli­cated in the in crease of the vacuolar pH, K+ accumulation, cell expansion and color transition from red to blue in petals 01 Ipomoea lricolor(Yoshida et al" 20091. Also, Arabidopsis nhx1 mutant plants had reduced leaf area and smaller epi­dermal ce lis (Apse et al., 20031, suggesting that these mutant plants do not supply the vacuoles with suffícient osmotic potential for the normal expansion of leaf cells, which largely depends on K+ accumulation (Maggio et al., 20061.

Under salt stress, K+ concentrations in different parts of tomato plants decreased, whereas the Na+ concentration increased in both transgenic and control lines, following well-known patterns of salt-related mineral changes in tomato (Rush and Epstein, 1981; Olias el al" 20091. However, no consistent differences in Na'" contents among transgenic and control lines were observed after saline treatments (Tables 1 and S1), despite the significant enhancement of Na+/H+ antiporter activity in tonoplast vesicles purified from these transgenic plants (Figure 1). By contrast, the expres­

sion of AtNHX1 led to the enhanced retention of K+ in transgenie Iines. The dual affinity 01 AtNHX1 lar Na· and K· (Figure 11 (Venema el al., 2002; Apse el al., 2003;Yamaguchi et al., 20051 and its Km for K· (21 mM, this work; 12.2 mM, Yamaguchi el al., 2005) imply that under regular growth conditions AtNHX1 mediates the uptake 01 K· into vacuoles. Under salinity stress, Na+ transport will also take place because of the rising concentration of this ion in the cytosol. However, the cytosolic concentration of K+ would still be higher than that of Na+ under most circumstances, including

when plants are growing under salinity stress {Carden et al., 2003; Tester and Davenport, 2003; Munns and Tester, 20081.

For instance, single-cell measurements of cytosolic activities of Na+ and K+ in the root cortex of two varieties of barley

subjected to severe salt stress combined with low external K' (200 mM NaCI and 0.1 mM K'I, showed that the Na'

activity in the cytosol ranged from 2 to 28 mM, depending on the plant variety and the duration of the treatment, whereas K+ the activity was 40-63 mM. By contrast, Na+ activity in vacuo les was 26-88 mM, which would require active trans­

port, i.e. against the electrochemical gradient of Na+, to compartmentalize cytosolic Na+ into the vacuole. In this

scenario, this likely occurs in the cytosol of many plants thriving under salt stress (Munns and Tester, 2008): K+jH+

exchange by NHX1-like proteins could predominate over, but not suppress, Na+jH+ exchange. The relative prevalence

of each exchange would be dependent on the selectivity of the particular NHX protein being considered, and the ionic environment in the cytosol, which in turn will be dependent

on the plant species and the intensity and duration of the salinity stress. In addition, Na+ transport across the tono­plast is bidirectional (Demidchik el al., 2002; Tester and

Davenport, 20031. Backllow lrom the vacuole to the cytosol,

probably through ion channels, demands sustained influx by Na+jH+ exchange to keep Na+ compartmentalized. Thus, even ifthe K+ : Na+ ratio in the cytosol is high, and the NHX1-like proteins do not discriminate between K+ and Na+, they can still contribute to significant Na+ compartmentation

provided that the 'Ieak' from the vacuole to the cytosol is low. These variables, and the conceivable diverse biochem­

ical behavior of NHX-type proteins isolated from various sources, and ectopically expressed in a range of plant species, could explain the disparate results reported regard­ing the enhanced accumulation of Na+, K+ or both, in the

transgenic plants overexpressing NHX proteins. To compli­cate matters further, Yamaguchi et al. (2005) have shown

thatthe ion selectivity 01 AtNHXl can be modulated through a regulatory mechanism involving binding of a calmodulin­

like protein to the e-terminal part of the transporter. This interaction resulted in a further gain of K+ selectivity over Na+, albeit these results were obtained in yeast and have not

been confirmed in planta. Interestingly, a genetic screen for

mutants of AtNHX1 that enhanced the salt tolerance ofyeast cells resulted in the isolation of protein variants with improved discrimination of K+ over Na+ (Hernandez et al.,

20091. Together, these data and the results shown here support the notion that significant transport of K+ into

vacuoles by AtNHX1 is an essential component of the salt tolerance conveyed by this exchanger. Another layer of regulation 01 the NHX proteins is imparted by the SOS pathway regulating the efflux and net content of Na+ in

plants. The tonoplast Na+jH+ exchange attributed to NHX

proteins in Arabidopsis was, when compared with the wild type, significantly higher, greatly reduced and unchanged in the 5051, 5052 and 5053 mutants, respectively (Oiu et al.,

20041. An activated lorm 01 the 5052 protein kinase added

in vitro increased tonoplast Na+jH+ exchange activity in

vesicles isolated from 5052, but did not have any effect on the activity in vesicles isolated from the wild type, 5051 or

5053. Moreover, 5052 interacts with and upregulates the transport activity of the vacuolar H+-ATPase (Batelli et al.,

2007). These results demonstrate that tonoplast Na+-K+jH+

exchange is a target ofthe SOS regulatory pathway, and that there may be coordinate regulation of ion transporters in the

tonoplast and plasma membrane. If NHX-type proteins can increase salt tolerance without

necessarily enhancing Na+ accumulation into vacuoles, the

question then remains ofwhat is the biochemical or cellular

mechanism that conveys halotolerance to the tomato plants overexpressing the AtNHX1 protein. We suggest that this tolerance derives from the significant role that the AtNHX1 antiporter plays in K+ homeostasis by capturing K+ in the

vacuoles, as well as the concurrent increment of compatible solutes (free sugars and proline) inthe cytosol.lt is generally accepted that Na+ inhibits K+ uptake and elicits K+ loss (Maathuis and Amtmann, 1999; Shabala el al., 20061. Con­sequently, the enhanced capacity of K+ uptake and subse­

quent retention is a critical adaptation to salinity stress (Maathuis and Amtmann, 1999; Borsani el al., 2001; Maggio et al., 2006). A negative correlation between Na+-triggered K+ loss and salt tolerance has been described previously (Chen el al., 2005; Shabala el al., 20061. Owing to the

enlarged K+ pool created before the onset of salinity stress,

and the subsequent retention afterwards, the transgenic plants would be better poised to withstand the imposition of salt stress, particularly il applied as a salt-shock (Figure 21. The larger vacuo lar pool of K+ could sustain and stabilize the

cytosolic K+ concentrations for longer periods oftime in the transgenic plants. Only later, as Na+ concentrations increase

within plant cells and the apoplast, and the cytosolic and vacuo lar pools of K+ are eventually depleted, would the

contribution of AtNHX1 to salt tolerance gradually decline, and salt-specific maladies appear (Munns, 2002). Greater K+

contents in salt-tolerant transgenic plants expressing NHX proteins have also been described by others (Xue et al.,

2004; Wu el al., 2005; Zhao el al., 2006; Rodriguez-Rosales

el al., 20081. In addition, the enlarged pool 01 K' in the vacuoles of tomato plants expressing AtNHX1 required the coordinated accumulation of compatible solutes (Table 3).

Transgenic AtNHX1 plants showed a significantly greater accumulation offree sugars even iftheywere nottransferred

to salt. Under salt stress, the transgenic plants accumulated more proline than controls, another feature often linked to salt tolerance (Hasegawa et al., 2000). However, the com­bined accumulation of K+ and compatible osmolytes in the

transgenic plants did not result in a significantly different osmotic potential compared with control plants (Table 3).

Therefore, we posit that the survival of AtNHX1-expressing plants under salt-shock was unlikely to be caused by improved osmotic adjustment, but instead by the greater

K+ accumulation befo re salt treatment and K+ retention

afterwards, together with the well-known protective effects of compatible solutes agai nst the dehydration and oxidative

stress associated with salinity. The additive effect of each of these individual features may contribute to improve the salt tolerance of the transgenics.

Concluding remarks

Our results reveal that NHX protei ns are li kely candidates for the H+-linked K+transport responsible for active K+ uptake at the tonoplast, to facilitate the partitioning of K+ between the

vacuole and the cytosol. This critical function should be taken into consideration for understanding the salt-tolerant

phenotype widely reported for plants overexpressing NHX­type proteins, some ofwhich are not significantly affected in Na+ compartmentation.

EXPERIMENTAL PROCEDURES

Tomato transformation and molecular methods

For plant transformation, the AtNHXl open reading frame {ORF) was inserted into vector pBI321 {Martínez-Atienza et al., 2007) between the CaMV 355 gene promoter and the nopaline synthase gene {NOS) terminator. The resulting plasmid was electroporated into Agrobacterium tumefaciens strain LBA4404 for transformation of cotyledon pieces. The protocol used was as described by Meissner et al. {1997), except that 150 mg mi-lar 60 mg ml-l

ticarcillin were used. In this study, only homozygous T2 and T3

plants {established by segregation analysis) oftransgenic lines were used in physiological or phenotypic experiments. Reverse tran­scription {RT)-PCR for the detection of gene transcripts was per­formed as described in Ruiz et al. {1998). The PCR reactions were performed with primers annealing to genes AtNHXl {AF106324), tomato HAK5 {OQ489721) and tomato TOM41 {U60480). For western blotting, microsomes were purified as described below and probed with antibodies raised against AtNHX1 {Quintero et al., 2000). The AtNHX1 protein was purified as described by Venema et al. {2002).

Plant culture and chemical analyses

Seeds were germinated at 28"C in Petri dishes, and 3-day-old seedlings were transplanted into plastic holders containing rock­wool, and were then transferred to 8-L plastic containers with modified Long Ashton solution, containing: 4 mM N0 3-, 1 mM H2P04-,3 mM SO/-, 4 mM K+, 2 mM Ca2 +,1 mM Mg2 +,50 11M Fe {as FeEDDHA), 10 11M Mn, 1 11M CU, 5 11M Zn and 30 11M B. In the standard medium, the K+ concentration was 4 mM. In experiments with different K+ levels, the required concentration was obtained by adding 0.5 M K2S04. Salinity treatments were administered to 2-week-old plants in hydroponic culture. Salt was added at 25 mM increments every 12 h until reaching 75 or 150 mM Na+ {as NaCI), or as a salt-shock with 300 mM Na+ {as Na2S04), in the nutrient solu­tion. Plants were kept in a growth chamber with the following conditions {day/night): 25/20 ± 2°C; 40/60% relative humidity; 14-h light; photosynthetically active radiation {PAR), 250 I1mol m-2 S-l. For ion analyses, plants were separated into leaves, stems {includ­ing petioles), fruits and roots, and were weighed. Roots were washed thoroughly in tap water and blotted dry befare weigh­ing. AII plant parts were dried at 70°C for 48 h in a forced-air oven. From fresh- and dry-weight measurements, water contents {in 9 H20 {g dry weight¡-l) were obtained. Potassium and sodium

were extracted from finely ground material with hot water under pressure by autoclaving samples, and then measured by atomic absorption spectrophotometry {AAS) {Perkin-Elmer 1100B, http:// www.perkinelmer.com). Sugar content was estimated by the phenol-sulfuric assay using glucose as standard {Ashwell, 1969). Proline was determined by reverse-phase HPLC following pre­column derivatization with phenylisothiocyanate {Heinrikson and Meredith, 1984). Osmotic potentials were determined from saps extracted from frozen tissues using a Wescor psychrometer HR-33T and sample chamber C-52. AII statistical analyses were performed with the software package STATISTIX {http://www.statistix.com).

Vacuolar membrane isolation and ion transport

Isolation of tonoplast membranes and measurement of cation/ proton exchange were as described by Barkla et al. {1999), except that fluorescence quenching of 9-amino-6-chloro-2-methoxy-acri­dine {ACMA) was used to monitor the formation and dissipation of pH gradients. Purified tonoplast vesicles {50 I1g of protein) were added to a buffer containing 250 mM mannitol, 10 mM bis­tris-p ropane-2-{ N-morph oli ne)-ethanesu Ip han ic acid {BTP-MES) {pH 8.0), 100 mM tetramethyl ammonium chloride, 3 mM MgS0 4 and 1 11M ACMA {1 mi final volume). Fluorescence was recorded with a Hitachi fluorescence spectrophotometer {FL-2500; http:// www.hitachi.com) in a thermostated cell {26°C) at excitation and emission wavelengths of 415 and 485 nm, respectively {slit width, 10 nm). The reaction mixture was stirred and maintained at 26°C throughout the transport assays. Formation of acid-inside pH gra­dients were started with the addition of 1.5 mM ATP-BTP {pH 8.0). When fluorescence stabilized, the initial rate of dissipation was measured after the addition of NaCI or KCI salts. Exchange rates are expressed as fluorescence recovery relative to that prior to ATP addition {f1F/Fmax) per minute and milligram of protein. To estimate the kinetic parameters of AtNHX1, the rates oftransport in wild-type vesicles were subtracted from the rates obtained for vacuoles con­taining AtNHX1. Exchange rates at each substrate concentration were fitted to Michaelis-Menten kinetics by using non linear regression with KALEIDAGRJ\PH {Synergy Software, http://www. synergy.com).

Root K+ uptake

Root K+ uptake was estimated by measuring Rb+ uptake {Benlloch et al., 1989) or measuring K+ depletion from the incubation solution with a K+-specific electrode. Tomato plants grown in full nutrient solution were transferred to K+ -free solution for 48 and 96 h befare measuring Rb+ uptake in nutrient media containing various con­centrations of RbCI for 10 mino Then, roots were immersed in ice­cold 5 mM CaS04 for 5 min, blotted on filter paper, cut off and their weight determined befare being frozen at -70°C. The Rb+ concen­tration was determined in centrifuged homogenates by AAS. Accumulation rates at each Rb+ concentration were fitted to Michaelis-Menten kinetics by using non linear regression with KALEIDAGRJ\PH {Synergy Software). For K+-uptake measurements using the K+-selective electrode, plants were transferred to a med­ium containing 0.25 mM KCI and 2 mM CaCI 2. Variation in K+ con­centration was continuously recorded using an ion meter equ ipped with K+ and reference electrodes {Crison). An additional estimate of K+ uptake was performed by AAS analysis of K+ concentration in the xylem sap collected from de-topped plants at harvest.

Vacuolar compartmentation

Vacuo lar contents in leaves and roots were analyzed by EDX. For EDX analysis, pieces of freshly halVested fully expanded leaves or the main root were used from plants grown in different salinity

levels. The plant pieces were mounted in slots of copper holders and fixed with optimal cutting temperature compound {BDH, http:// uk.vwr.com). The copper holder containing the samples was dipped into a bath of slush N2 before transferring it under vacuum into the cryopreparation chamber {CT1500; Oxford Instruments, http:// www.oxford-instruments.com) attached to the SEM {DSM 960; Zeiss, http://www.zeiss.com). The temperature in the chamber was left to rise from -163 to -90°C and was then set for 10 min for ice sublimation before sputter-coating with gold {2 min). The SEM was fitted with an ATW detector interfaced with a Link ISIS analyzer {http://www.oxford-instruments.com). Samples were analyzed keeping the following parameters constant: accelerating voltage, 15 kV; take-off angle 42°; collecting time of X-ray counts, 100 s; working distance between sample and detector, 24 mm. Measure­ments were performed by focusing on exposed vacuo les of specific ce lis {mesophyll parenchyma in leaves, or cortical and vascular cylinder cells in roots), or covering cell groups from different tissues in roots {cortex and vascu lar cylinder). Solutions containing known ion concentrations and plant extracts obtained from the remaining parts of leaves and roots, in which ion concentration was deter­mined by AAS, were used to determine the response ofthe analyzer to K+ and Na+. The linear correlation between actual element concentration {mM) and EDX recordings {counts) was statistically significant {K+, (== 0.68, P< 0.05; Na+, (== 0.85, P< 0.01).

Electrophysiological experiments

For electrophysiological experiments, tomato seeds were germi­nated in distilled water and transferred for 3-4 days to media con­taining 2 mM CaCI2 and 0.1 mM KCI. Membrane potentials {Em ) were measured in epidermal root cells oftomato seedlings, as described previously {Rubio et al., 2004). Diffusion potentials {Eo) were reached after the addition of 1 mM NaCN and 1 mM SHAM {Fern­ández et al., 1999) to the aboye assay medium, buffered with 9 mM BTP-MES, pH 6.0. Cytosolic K+ activities were determined by two different approaches. First, diffusion potentials were measured at increasing external K+ concentrations {1, 10, 25 and 50 mM KCI) with single-barrel microelectrodes. Different diffusion potentials in con­trol and transgenic roots indicated unequal cytosolic K+ activities {Mith6fer et al., 2005). Second, cytosolic K+ activities were directly measured by double-barreled K+-selective microelectrodes. The microelectrode pre-treatment and backfilling was similar to the W­or Na+-selective microelectrodes described previously {Fernández et al., 1999; Rubio et al., 2005). The microelectrodes were filled with a K+-sensor cocktail containing potassium ionophore I {cocktail B, cat. no. 60398; Fluka, now part of Sigma-Aldrich, http://www.sig­maaldrich.com) dissolved in a mixture of polyvinylchloride!tetra­hydrofuran {40 mg ml-') at a ratio 30/70 {v/v) {Mith6fer et al., 2005). The signals from the K+-selective and voltage barreis were recorded and simultaneously subtracted by a high-impedance differential amplifier {FD223; World Precision Instruments, http://www.wpi­inc.com). The difference was calibrated before and after the exper­iments with different KCI solutions {from 1 to 500 mM KCI) maintained at a constant ionic strength by the addition of MgCI 2

{Walker et al., 1995). Calibration curves showed slopes around 51 mV/pK+. The impalements were stable for at least 30 mino

ACKNOWLEDGEMENTS

We are indebted to Bronwyn Barkla and Rosario Herrera-Estrella {Instituto de Biotecnologia, UNAM, Cuernavaca, México) and Andrés Belver {EEZ, CSIC, Granada, Spain) for their helpful advice in the meas u rement of antiporter activity. This work was supported by grants BFU2006-06968 from 'Ministerio de Educación y Ciencia' and AGR-1482 from 'Junta de Andalucía' to JMP. Additional support

was obtained from CSIC-CONACYT Bilateral Cooperation Grant 2004MX0021.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version ofthis article: Figure S1. Transgene expression. Table S1. Average fresh weight, K+ and Na+ concentrations, and fruit production of salt-treated plants. Table S2. Potassium concentration in leaf and roots in the wild type and four transgenic lines. Table S3. Potassium concentration in leaves grown under different regimes of K+ availability. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information {other than missing files) shou Id be addressed to the authors.

REFERENCES

Apse, M.P., Aharon, G.S., Snedden, W.A. and Blumwald, E. (1999) Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science, 285,1256-1258.

Apse, M.P., Sottosanto, J.B. and Blumwald, E. (2003) Vacuolar cation/H+ exchange, ion homeostasis, and leaf development are altered in a T-DNA insertional mutant of AtNHX1, theArabidopsis vacuolar Na+/H+ antiporter. Plant J. 36, 229-239.

Ashwell, G. (1969) New colorimetric methods of sugar analysis. In Methods in Enzymology (Neufeld, E.F. and Ginsburg, V., ed). New York: Academic Press Inc, pp. 85-95.

Barkla, B.J., Vera-Estrella, R., Maldonado-Gama, M. and Pantoja, O. (1999) Abscisic acid induction of vacuolar H+-ATPase activity in Mesembryan­themum crystallinum is developmentally regulated. Plant Physiol. 120, 811-819.

Batelli, G., Verslues, P.E., Agius, F., Oiu, O., Fujii, H., Pan, S., Schumaker, I<.S., Grillo, S. and lhu, J.K. (2007) SOS2 promotes salt tolerance in part by interacting with the vacuolar H+-ATPase and upregulating its transport activity. Mol. Cell. Biol. 27,7781-7790.

Benlloch, M., Moreno, l. and Rodriguez-Navarro, A. (1989) Two Modes of Rubidium Uptake in Sunflower Plants. Plant Physiol. 90, 939-942.

Blumwald, E., Aharon, G.S. and Apse, M.P. (2000) Sodium transport in plant cells. Biochim. Biophys. Acta-Biomembranes, 1465, 140-151.

Borsani, O., Cuartero, J., Fernandez, J.A., Valpuesta, V. and Botella, M.A. (2001) Identification of two loci in tomato reveals disti nct mecha nisms for salt tolerance. Plant Cel/, 13,873-887.

Brett, C.l., Oonowitz, M. and Rao, R. (2005) Evolutionary origins of eukaryotic sodium/proton exchangers. Am. J. Physiol. Cell Physiol. 288, C223-C239.

Carden, O.E., Walker, O.J., Flowers, T.J. and Miller, A.J. (2003) Single-cell measurements of the contributions of cytosolic Na+ and K+ to salt toler­ance. Plant Physiol. 131,676-683.

Chen, l., Newman, l., lhou, M., Mendham, N., lhang, G. and Shabala, S. (2005) Screening plants for salt tolerance by measuring K+ flux: a case study for barley. Plant Cell and Enviran. 28, 1230-1246.

Oemidchik, V., Oavenport, R.J. and Tester, M. (2002) Nonselective cation channels in plants. Annu Rev Plant Biol. 53, 67-107.

Fernández, J.A., Garcia-Sanchez, M.J. and Felle, H.H. (1999) Physiological evidence for a proton pump and sodium exclusion mechanisms at the plasma membrane ofthe marine angiosperm Zastera marina L. J. Exp. Bot. 50,1763-1768.

Fukuda, A., Nakamura, A., Tagiri, A., Tanaka, H., Miyao, A., Hirochika, H. and Tanaka, Y. (2004) Function, intracellular localization and the importance in salt tolerance of a vacuolar Na+/H+ antiporter from rice. Plant Cell Physiol. 45,146-159.

Fukuda-Tanaka, S., Inagaki, Y., Yamaguchi, T., Satio, N. and lida, S. (2000) Colour-enhancing protein in blue petals. Nature, 407, 581.

Gaxiola, R.A., Rao, R., Sherman, A., Grisafi, P., Alper, S.l. and Fink, G.R. (1999) The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast. Prac. Natl. Acad. Sci. USA, 96, 1480-1485.

Hasegawa, P.M., Bressan, R.A., lhu, J.K. and Bohnert, H.J. (2000) Plant cel­lular and molecular responses to high salinity. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 463-499.

Heinrikson, R.l. and Meredith, S.C. (1984) Amino acid analysis by reverse­phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate. Anal. Biochem. 136,65-74.

Hernandez, A., Jiang, X., Cubero, B., Nieto, P.M., Bressan, R.A., Hasegawa, P.M. and Pardo, J.M. (2009) Mutants of the Arabidopsis thaliana cation/H+ antiporter AtNHX1 conferring increased salt tolerance in yeast: the endo­some/prevacuolar compartment is a target for salt toxicity. J. Biol. Chem. 284,14276-14285.

leigh, R.A. (2001 ) Potassium homeostasis and membrane transport. J Plant Nutr. Soil Sci. 164, 193-198.

Maathuis, F.J.M. and Amtmann, A. (1999) K+ nutrition and NM toxicity: the basis of cellular K+/NM ratios. Ann. Bot. 84, 123-133.

Maggio, A., lhu, J.K., Hasegawa, P.M. and Bressan, R.A. (2006) Osmogenet­ics: Aristotle to Arabidopsis. Plant Cel/, 18, 1542-1557.

Martínez-Atienza, J., Jiang, X.Y., Garciadeblas, B., Mendoza, l., lhu, J.K., Pardo, J.M. and Ouintero, F.J. (2007) ConselVation of the salt overly sen­sitive pathway in rice. Plant Physiol. 143, 1001-1012.

Martinoia, E., Massonneau, A. and Frangne, N. (2000) Transport processes of solutes across the vacuolar membrane of higher plants. Plant Cell Physiol. 41,1175-1186.

Meissner, R., Jacobson, Y., Melamed, S., levyatuv, S., Shalev, G., Ashri, A., Elkind, Y. and levy, A. (1997) A new model system for tomato genetics. PlantJ. 12, 1465-1472.

Mithofer, A., Ebel, J. and Felle, H.H. (2005) Cation fluxes cause plasma membrane depolarization involved in beta-glucan elicitor-signaling in soybean roots. Mol. Plant Micrabe Interact. 18,983-990.

Munns, R. (2002) Comparative physiology of salt and water stress. Plant Cell and Enviranment, 25, 239-250.

Munns, R. and Tester, M. (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol. 59, 651-681.

Nieves-Cordones, M., Martinez-Cordero, M.A., Martinez, V. and Rubio, F. (2007) An NH4 + -sensitive component dominates high-affinity K+ uptake in tomato plants. Plant Sci. 172,273-280.

Nieves-Cordones, M., Miller, A., Alemán, F., Martínez, V. and Rubio, F. (2008) A putative role for the plasma membrane potential in the control of the expression of the gene encoding the tomato high-affinity potassium transporter HAK5. Plant Mol. Biol. 68, 521-532.

Ohnishi, M., Fukada-Tanaka, S., Hoshino, A., Takada, J., Inagaki, Y. and lida, S. (2005) Characterization of a novel Na +/W antiporter gene InNHX2 and comparison of InNHX2with InNHX1, which is responsible for blue flower coloration by increasing the vacuolar pH in the Japanese morning glory. Plant Cell Physiol. 46, 259-267.

Ohta, M., Hayashi, Y., Nakashima, A., Hamada, A., Tanaka, A., Nakamura, T. and Hayakawa, T. (2002) Introduction of a NM!f-h antiporter gene from Atriplex gmelini confers salt tolerance to rice. FEBS Lett 532, 279-282.

Olias, R., Eljakaoui, l., li, J., Alvarez De Morales, P., Marin-Manzano, M.C., Pardo, J.M. and Belver, A. (2009) The plasma membrane NM!H+ antiporter SOS1 is essential for sa It tolera nce i n tomato a nd affects the pa rtitioni ng of NM between plant organs. Plant Cell & Enviran. 32, 904-916.

Pardo, J.M., Cubero, B., leidi, E.O. and Ouintero, F.J. (2006) Alkali cation excha ngers: roles in cell ula r homeostasis a nd stress tolera nce. J. Exp. Bot. 57,1181-1199.

Oiu, O.S., Guo, Y., Ouintero, F.J., Pardo, J.M., Schumaker, K.S. and lhu, J.K. (2004) Regulation ofvacuolar NM!H+ exchange in Arabidopsis thaliana by the salt-overly-sensitive (SOS) pathway. J. Biol. Chem. 279, 207-215.

Ouintero, F.J., Blatt, M.R. and Pardo, J.M. (2000) Functional conservation between yeast and plant endosomal NM/H+ antiporters. FEBS Lett. 471, 224-228.

Rodriguez-Navarro, A. (2000) Potassium transport in fungi and plants. Bio­chim. Biophys. Acta-Rev. Biomembranes, 1469, 1-30.

Rodriguez-Rosales, M.P., Jiang, X., Galvez, F.J., Aranda, M.N., Cubero, B. and Venema, K. (2008) Overexpression ofthe tomato K+/H+ antiporter LeNHX2 confers salt tolerance by improving potassium compartmentalization. New Phytol. 179, 366-377.

Rubio, l., Rosado, A., linares-Rueda, A., Borsani, O., Garcia-Sanchez, M.J., Valpuesta, V., Fernandez, J.A. and Botella, M.A. (2004) Regulation of K+ transport in tomato roots by the TSS1 locus. Implications in salt tolerance. Plant Physiol. 134,452-459.

Rubio, l., linares-Rueda, A., Garcia-Sanchez, M.J. and Fernandez, J.A. (2005) Physiological evidence for a sodium-dependent high-affinity phosphate and nitrate transport at the plasma membrane of leaf and root cells of Zastera marina L. J. Exp. Bot. 56, 613-622.

Ruiz, M.T., Voinnet, O. and Baulcombe, D.C. (1998) Initiation and maintenance of vi rus-i nduced gene si lenci ng. Plant Cell, 10, 937-946.

Rush, D.W. and Epstein, E. (1981) Comparative Studies on the Sodium, Potassium, and Chloride Relations of a Wild Halophytic and a Domestic Salt-Sensitive Tomato Species. Plant Physiol. 68, 1308-1313.

Shabala, S., Demidchik, V., Shabala, l., Cuin, T.A., Smith, S.J., Miller, A.J., Davies, J.M. and Newman, I.A. (2006) Extracellular Ca2 + ameliorates NaCI-induced K+ loss from Arabidopsis root and leaf cells by controlling plasma membrane K+-permeable channels. Plant Physiol. 141,1653-1665.

Tester, M. and Davenport, R. (2003) NM tolerance and NM transport in higher plants. Ann. Bot. 91, 503-527.

Venema, K., Ouintero, F.J., Pardo, J.M. and Donaire, J.P. (2002) The Arabid­opsis NM!H+ exchanger AtNHX1 catalyzes low affinity NM and K+ trans­port in reconstituted liposomes. J. Biol. Chem. 277, 2413-2418.

Venema, K., Belver, A., Marin-Manzano, M.C., Rodriguez-Rosales, M.P. and Donaire,J.P. (2003) A novel intracellular K+/H+ antiporter related to NM/H+ antiporters is important fo r K+ ion homeostasis in plants. J. Biol. Chem. 278,22453-22459.

Walker, D.J., Smith, S.J. and Miller, A.J. (1995) Simultaneous measurement ofintracellular Ph and K+ or N03- in barley root-cells using triple-barreled, ion-selective microelectrodes. Plant Physiol. 108,743-751.

Walker, D.J., leigh, R.A. and Miller, A.J. (1996) Potassium homeostasis in vacuolate plant cells. Proc. Natl Acad. Sci. USA, 93,10510-10514.

Wu, Y.Y., Chen, O.J., Chen, M., Chen, J . and Wang, X.C. (2005) Salt-tolerant transgenic perennial ryegrass (Lolium perenne L.) obtained by Agrabac­terium tumefaciens-mediated transformation of the vacuolar Na+/H+ anti­porter gene. Plant Sci. 169,65-73.

Wu, C., Gao, X., Kong,X., lhao, Y. and lhang, H. (2009) Molecular cloning and functiona I a na Iysis of a Na+/H+ a ntiporter gene ThN HX1 from a ha lophytic plant Thellungiella halophila. Plant Mol. Biol. Rep. 27, 1-12.

Xue, l.Y., lhi, D.Y., Xue, G.P., lhang, H., lhao, Y.X. and Xia, G.M. (2004) Enhanced salt tolerance of transgenic wheat (Tritivum aestivum L.) expressing a vacuolar NM/H+ antiporter gene with improved grain yields in saline soils in the field and a reduced level of leaf NM. Plant Sci. 167, 849-859.

Yamaguchi, T., Aharon, G.S., Sottosanto, J .B. and Blumwald, E. (2005) Yac­uolar NM/H+ antiporter cation selectivity is regulated by calmodulin from within the vacuole in a Ca2+- and pH-dependent manner. Prac. Natl Acad. Sci. USA, 102, 16107-16112.

Yamaguchi, T., Fukuda-Tanaka, S., Inagaki, Y., Saito, N., Yonekura­Sakakibara, K., Tanaka, Y., Kusumi, T. and lida, S. (2001) Genes encoding the vacuolar Na+/Ha+ exchanger and flower coloration. Plant Cell Physiol. 42, 451-461.

Yokoi, S., Ouintero, F.J., Cubero, B., Ruiz, M.T., Bressan, R.A., Hasegawa, P.M. and Pardo, J.M. (2002) Differential expression and function of Arabidopsis thaliana NHX NM/H+ antiporters in the salt stress response. Plant J. 30, 529-539.

Yoshida, K., Kawachi, M., Mori, M., Maeshima, M., Kondo, M., Nishimura, M. and Kondo, T. (2005) The involvement of tanoplast proton pumps and NM(K+)/H+ exchangers in the change of petal color during flower opening of morning glory, Ipomoea tricolor cv. Heavenly Blue. Plant and Cell Physiology, 46, 407.

Yoshida, K., Miki, N., Momonoi, K., Kawachi, M., Katou, K., Okazaki, Y., Uozumi, N., Maeshima, M. and Kondo, T. (2009) Synchrony between flower opening and petal-color change from red to blue in morning glory, lpomoea tricolor cv. Heavenly Blue. Prac. Jpn.Acad., Ser. B, Phys. Biol. Sci. 85,187-197.

lhang, H.X. and Blumwald, E. (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nat. Biotechnol. 19, 765-768.

lhao, F.Y., lhang,X.J., li, P.H., lhao, Y.X. and lhang, H. (2006) Co-expression of the Suaeda salsa SsNHX1 and Arabidopsis AYP1 confer greater salt tolerance to transgenic rice than the single SsNHX1. Mol. Breeding, 17, 341-353.